Our studies have proceeded along three lines: (1) a functional analysis of the partition module of plasmid prophage P1. This is a set of genes that ensure the distribution of plasmid replicas to daughter cells such that no cell is deprived of a plasmid copy. Bacterial homologs have been recently recognized in both gram-positive and gram-negative bacteria, of which some have been shown to be functional in partitioning too. (2) a study of a diminutive partition module from the pTAR plasmid of Agrobacterium tumefaciens and (3) an analysis of a P1- encoded failsafe mechanism (dubbed plasmid addiction) responsible for killing cells that have lost the plasmid. (1). We have recently demonstrated, by reversible in vivo cross-linking, that ParB, the centromere-binding protein of P1, is able to spread outward along the DNA from a nucleation site in the plasmid centromere, silencing genes in its path. The putative polymer may extend up to 8 or 9 kb from its nucleation site under normal physiological conditions. Its spread can be halted by a repressor-operator complex in its path. An RNA polymerase heading towards the nucleation site appears less vulnerable to being silenced than one oppositely oriented. Genetic evidence suggests that ParB spreading beyond the centromere proper may be required in the partitioning process, an hypothesis that we are subjecting to a more rigorous physiological test. ParB spreading and the silencing it entails offer a new handle on the partitioning process as does the advent of GFP-fusion techniques that permit cytological studies on living cells in real time. We will channel the limited resources of the laboratory to exploit this new knowledge. (2) Components of the smallest known plasmid partition module have been characterized with an aim to determining the essential conserved features of partition proteins and their cognate centromeres. We expect shortly to submit a manuscript for publication on this topic, but do not plan to continue work with this material, although we would be pleased to have others continue this research effort. (3). Having completed studies of the autogenous regulation of the P1 plasmid addiction operon, we have undertaken purification of the addiction proteins (a toxin and its antidote) for structural analysis by NMR (with S. Roy, Bose Institute). Physiological and biochemical experiments indicate that the target of the toxin is protein synthesis and that the antidote is capable of reversing the inhibitory effect of the toxin. Efforts directed towards defining the step in protein synthesis that is affected will be continued by Roy Magnuson, the post- doctoral fellow who has been pursuing this project here. He is now Assistant Professor at the University of Alabama, Huntsville. Finally, in collaboration with the groups of M. Lobocka (Warsaw) and F. C. Blattner (Wisconsin), we are planning on publishing an extensively annotated complete nucleotide sequence of P1. Yarmolinsky has also agreed to prepare reviews on Bacterial Silencing and on Plasmid and Chromosome Partitioning in Bacteria for publication in 2000 and 2001 respectively. References1. O. Rodionov, M. Lobocka and M. Yarmolinsky, Silencing of genes flanking the P1 plasmid centromere, Science, 283: 546-9 (1999).2. K. Kalnin, S. Stegalkina and M. Yarmolinsky, in preparation.3. R. Magnuson and M. Yarmolinsky, Corepression of the P1 addiction operon by Phd and Doc, J. Bacteriol., 180:6342-51 (1998).4. M. Lobocka, D. Rose, M. Rusin, A. Samojedny, M. Yarmolinsky and F.C. Blattner, Complete nucleotide sequence of P1, a bacteriophage that lysogenizes as a plasmid: Analysis based on two related isolates, in preparation. - Bacterial genetics, DNA binding proteins, partition, Plasmids, toxins, - Neither Human Subjects nor Human Tissues